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| United States Patent |
5,561,108
|
|
Tsay
, et al.
|
October 1, 1996
|
Preparation of .alpha..sub.1 -antichymotrypsin
Abstract
Human .alpha..sub.1 -antichymotrypsin (ACT) can be purified from solutions
containing human .alpha..sub.1 -proteinase inhibitor (PI) and antithrombin
III (AT-III) using chromatography adsorption steps at carefully controlled
pH and conductivity. The separated ACT retains in vitro inhibitory
capacity and has potential therapeutic use.
| Inventors:
|
Tsay; Grace C. (Walnut Creek, CA);
Cheung; Neal K. H. (Vallejo, CA);
Bettencourt; Jeffrey D. (Walnut Creek, CA)
|
| Assignee:
|
Bayer Corporation (Elkhart, IN)
|
| Appl. No.:
|
282860 |
| Filed:
|
July 29, 1994 |
| Current U.S. Class: |
514/12; 530/380; 530/395; 530/415; 530/416; 530/830 |
| Intern'l Class: |
C07K 001/18; C07K 001/22; C07K 014/47; A61K 038/17 |
| Field of Search: |
530/380,395,412,415,416,829,830,831
514/12
435/183
|
References Cited
U.S. Patent Documents
| 4379087 | Apr., 1983 | Coan et al. | 530/380.
|
| 5079336 | Jan., 1992 | Rubin et al. | 530/350.
|
Other References
Travis et al. Biochemistry 17(26) 5647-5651 1978.
Laine et al. Biochim Biophys Acta 668 429-438 1981.
Laine et al. Eur. J. Biochem 197 209-215 1991.
Laine et al Clin Chim Acta 190 163-174 1990.
Abdullah et al. Arch Biochem Biophys. 225(1) 306-312 1983.
Siddiqui et al Biochem Biophys Res. Commun. 95(4) 1737-1742 1980.
|
Primary Examiner: Fleisher; Mindy
Assistant Examiner: Degen; Nancy J.
Attorney, Agent or Firm: Giblin; James A.
Claims
We claim:
1. A method of preparing human alpha-1-antichymotrypsin (ACT) from Cohn
Fraction IV-1 paste which includes human ACT and human alpha.sub.1
proteinase inhibitor (PI) and human anti-thrombin III (AT-III) comprising
the steps of
(A) obtaining an aqueous solution of a Cohn Fraction IV-1 paste that
includes ACT, PI and AT-III;
(B) contacting the solution with an anion exchange resin at a conductivity
of about 1.0 to 2.5 mmho/cm and a pH of about 6.5 to adsorb the ACT, PI
and AT-III;
(C) washing the resin under conditions of pH of about 6.5 and conductivity
of 7.0 to 7.8 mmho/cm to elute substantially all of the PI and AT-III;
(D) eluting ACT from the resin to form an eluate solution;
(E) contacting the eluate solution at a pH of about 6.8 and a conductivity
of 1.0 to 2.0 mmho/cm with DNA-cellulose to adsorb the ACT; and
(F) eluting the ACT from the DNA-cellulose.
Description
BACKGROUND OF THE INVENTION
1. Field
This disclosure relates generally to the preparation of human blood plasma
proteins and specifically with the preparation of alpha.sub.1
-antichymotrypsin (ACT) from a mixture of ACT, alpha.sub.1 -proteinase
inhibitor (PI) and antithrombin III (AT-III) such as that found in Cohn
Fraction IV-1 paste suspension obtained from human plasma.
2. Prior Art
Before the Second World War, scientists and physicians discovered that
human plasma could be used in blood replacement therapy. During the War,
difficulties of supply and storage of whole blood and plasma meant
battlefield shock could not be treated as effectively as possible. The
need for plasma proteins which could be stored and used on the battlefield
led Cohn and others (U.S. Pat. No. 2,390,074 (1945) and the J. Amer. Chem.
Soc., 68; p459 (1946)) to discover that proteins present in plasma could
be fractionated by selective precipitation in the presence of
water-soluble organic solvents or neutral salts. For a review of plasma
fractionation see, "The Plasma Proteins", Second Edition, Volume III, pp
548-550, Academic Press, New York, N.Y. (1977). The concentrated protein
mixtures could then be introduced into patients as needed. For example, if
excess bleeding was the problem, the physician could inject a
fibrinogen-enriched fraction. If the patient suffered burns or other
traumatic injury where the loss of plasma exceeded that of red blood
cells, the physician could use albumin, the colloid-osmotic regulator of
plasma.
In Cohn fractionation, ethanol is added to plasma and the pH is lowered at
sub-zero temperatures to selectively precipitate protein. After the
precipitate is separated from the supernatant, the pH of the supernatant
is lowered, and/or more alcohol is added to precipitate another fraction.
Today, this method of fractionation is still being used to separate
biologically active proteins that possess certain therapeutic qualities.
For instance, Factor VIII or anti-hemophilic factor is useful against
hemophilia; plasminogen, a precursor of plasmin, is used in the treatment
of acute thromboembolic disorders; gamma globulins, including immune serum
globulin and intravenous gamma globulin, are employed in the treatment of
congenital gamma globulin deficiency, measles, poliomyelitis and hepatitis
A and B; fibronectin has been identified as active in treatment of burns,
shock, cancer, etc.; anti-thrombin III is a coagulation inhibitor;
cryoprecipitate itself may be used directly for classic hemophilia; Plasma
Protein Fraction and albumin are useful in treatment of shock due to
burns, crushing injuries, abdominal emergencies, and any other trauma
producing a predominant loss of plasma fluids but not red cells; and
.alpha..sub.1 -proteinase inhibitor can be employed in the treatment of
emphysema.
Human .alpha..sub.1 -antichymotrypsin (ACT) is a serine protease inhibitor
that has, until now, only been isolated from human plasma or serum.
Although the precise biological function of ACT has not yet been
determined, it appears to be a multifunctional protein and its use for
various therapies has been suggested. (See, for example, U.S. Pat. No.
5,008,242 to J. Lezdey, et al.)
There is evidence to indicate that an important function of ACT is the
inhibition of proteases, such as chymotrypsin-like protease, mast cell
chymase, leukocyte cathepsin G (see Beatty, K., Bieth, J., Travis, J.:
Kinetic of association of serine proteinases with native and oxidized
.alpha..sub.1 -proteinase inhibitor and .alpha..sub.1 -antichymotrypsin,
J. Biol. Chem. 1980; 255:3931-3934) and pancreatic elastase (see Laine,
A., Davril, M. Rabaud, M., et al.: Human serum .alpha..sub.1
-antichymotrypsin is an inhibitor of pancreatic elastases, Eur. J.
Biochem. 1985; 151:327-331 and Davril, M., Laine, A., Hayem, A.: Studies
on the interactions of human pancreatic elastase 2 with human
.alpha..sub.1 -proteinase inhibitor and .alpha..sub.1 -antichymotrypsin,
Biochem. J. 1987; 245:699-704).
The biological properties of intact, cleaved and complexed forms of ACT
indicate the proteolytic-character of the protein may have a potential
role for therapeutic use in regulating infectious disease, pancreatitis,
lung disease and skin inflammation (see Rubin, H.: The biology and
biochemistry of antichymotrypsin and its potential roles as a therapeutic
agent, Biol. Chem. Hoppe-Sayler 1992; 373(7):497-502).
J. Travis et al. purified ACT to homogeneity from a human plasma pool
(Travis, J., Garner, D., Bowen, J.: Human .alpha..sub.1 -antichymotrypsin:
purification and properties, Biochemistry 1978; 17:5647-5651).
T. Katsunuma and his colleagues purified a DNA-binding protein, thought to
be a tumor marker, to homogeneity (Katsunuma, T. et al.: Purification of a
serum DNA binding protein (64DP) with a molecular weight of 64,000 and its
diagnostic significance in malignant diseases, Biochem. and Biophys. Res.
Comm., 93(2):552-557 (1980)). Later, the DNA-binding protein was found to
be ACT. Katsunuma used human serum as the starting material. They first
eluted 64DP from DEAE Sephadex with 225 mM NaCl. After dialysis, the 64DP
was further purified on DNA Cellulose. Finally to achieve homogeneity,
64DP was precipitated with ammonium sulfate and separated from
contaminating proteins on a size exclusion column.
In addition to isolation from whole plasma, human ACT has been cloned,
sequenced and expressed in Escherichia coli (see Rubin, H. et al.:
Cloning, expression, purification and biological activity of recombinant
native and variant human antichymotrypsins, J. Biol. Chem. 1990;
265:1199-1207). Rubin et al. used a Sepharose Fast Q column to separate
ACT activity from the crude bacterial lysate. The partially purified ACT
was then adsorbed to DNA Cellulose and eluted in 350-400 mM KCl.
Because of the potential uses of ACT, there is now a need for more
efficient ways of preparing large quantities of ACT from human plasma
especially from plasma fractions that also include varying amounts of PI
and AT-III.
Unexpectedly, we discovered that ACT can be purified from Cohn Fraction
IV-1. Fraction IV-1 contains many different proteins and difficulties in
separation preclude most commercial uses of this plasma fraction.
Isolation of ACT would therefore be a beneficial use of this otherwise
underutilized and normally discarded plasma fraction.
Compared to human serum, Cohn Fraction Paste IV-1 contains higher
concentrations of proteins, such as PI and AT-III, that are closely
related to ACT and would be expected to co-purify with ACT. During the
fractionation process to IV-1 paste, ACT is only concentrated 1-2 fold,
while PI is concentrated 3-10 fold and AT-III is concentrated 2-3 fold.
The concentrations of the proteins in the IV-1 paste are 30 mg PI/g paste,
5 mg AT-III/g paste and 5-10 mg ACT/g paste. Considering the difficulty in
separating ACT from these other proteins and its status as a minor
component, it would be considered unlikely that Fraction IV-1 paste would
be a suitable source of ACT. Surprisingly, we were able to isolate 2-3 g
ACT at a purity .gtoreq.90% from approximately 2.9 kg of IV-1 paste using
a modified form of the procedure of Katsunuma, et al.
In addition to its demonstrated use an as in vitro serine protease
inhibitor (e.g., it can be used as a reagent for PI studies), we have
proposed how ACT may be of potential therapeutic use. Details of our
discoveries are disclosed below.
SUMMARY OF THE INVENTION
Our method of preparing human alpha.sub.1 -antichymotrypsin (ACT) from a
solution which also includes alpha.sub.1 -proteinase inhibitor (PI) and
antithrombin III (AT-III) comprises the steps of
(A) contacting the solution containing ACT with an ion exchange resin at a
conductivity (ionic strength) and at pH sufficient to adsorb ACT;
(B) washing the adsorbent using a solution having pH, and conductivity
sufficient to remove substantially all PI and AT-III while not removing
the adsorbed ACT; and
(C) eluting the ACT from the resin.
In one preferred embodiment, step (C) is followed by further purification
steps of contacting the eluate solution of step (C) with DNA-cellulose
under conditions and pH sufficient to adsorb ACT and then elute the ACT
from the DNA cellulose.
In another preferred embodiment, the ACT is prepared from a suspension of
Cohn Fraction IV-1 paste which includes an aqueous solution of the ACT, PI
and AT-III having a conductivity of 1.0-2.5 mmho/cm and a pH of about 6.45
to 6.55. The ACT is purified using step (A) by chromatography on an anion
exchange resin (i.e., DEAE-Sepharose.RTM.) at about pH 6.5 followed by
washing the DEAE-Sepharose.RTM. with a wash solution having a pH of about
6.5 and a conductivity of 7.4 to 7.8 mmho/cm and eluting the ACT at a pH
of about 6.5 and a conductivity of 9 to 10.5 mmho/cm and, finally, by
adsorption to DNA-cellulose at pH 6.8 prior to final elution of the ACT.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 is a generalized flow chart showing how the preferred starting
material (Cohn fraction IV-1 paste suspension) is fractionated from human
plasma. FIG. 2 is a flow chart showing the preferred steps of our ACT
purification process.
MATERIALS AND METHODS
Purified .alpha..sub.1 -antichymotrypsin, .alpha..sub.1 -antiproteinase
inhibitor and antithrombin III from human plasma, rabbit antibodies to
human ACT and human neutrophil cathepsin G were purchased from Athens
Research, Athens, Ga. DNA-cellulose (5 ml) was purchased from Pharmacia,
Inc. Native calf thymus DNA and cellulose scale-up process DNA-cellulose
preparation, succinyl-ala-ala-pro-phe-p-nitroanilide and bovine
chymotrypsin were purchased from Sigma Chemical Co., St. Louis, Mo. Cohn
Fraction IV-1 was obtained from Miles Inc. DEAE-Sepharose.RTM. is a
registered trademark of Pharmacia, Inc. for its beaded agarose anion
exchange material.
DNA-Cellulose Preparation for Scale-Up Process
DNA-cellulose (3 L) was prepared according to the method of Litman (see
Litman, R. M.: A deoxyribonucleic acid polymerase from Micrococcus lutens
(Micrococcus hysodeikticus) isolated on deoxyribonucleic acid-cellulose,
J. Biol. Chem. 1968; 243:6222-6233). Cellulose (1 kg) was washed for 10
minutes with 1N HCL (15 L), rinsed with water and dried by air. Native
calf thymus (16 g) was mixed with cellulose (1 kg) in 8 L of 10 mM NaCL.
The lumpy mixture was spread out and dried under a blower for 3-5 hours
and finally allowed to stand overnight in the open. The dry matrix was
crushed and resuspended in ethanol. The slurry was then irradiated by a UV
lamp to form crosslinks to the cellulose.
Purification of Human ACT
Cohn Fraction IV-1 paste prepared generally in accordance with the
fractionation method of Cohn et al., J. Amer. Chem. Soc. 1948;68:459,
including ACT, PI and AT-III, was suspended in 24 volumes of 0.01M Tris pH
9.3 and heated at 40.degree. C. for one to one and a half hours. The
fraction IV-1 paste suspension was adjusted to pH 6.5 (70 liters) and was
loaded on a 30 kg DEAE-Sepharose.RTM. column. The DEAE column was
equilibrated with one column volume of 25 mM NaKPO.sub.4 pH 6.5 and washed
overnight with more than 10 column volumes of 75 mM NaKPO.sub.4 pH 6.5, 1
mM EDTA, to give a stable baseline. The partially purified ACT was eluted
with 3-5 volumes of 100 mM NaKPO.sub.4 pH 6.5. The 100 mM NaKPO.sub.4
eluate was ultrafiltered/diafiltered against 0.01M K.sub.2 HPO.sub.4 pH
6.8 (conductivity 1-2 mmho/cm) in a PM-10 3 ft..sup.2 cartridge and batch
contacted with the DNA-cellulose (3 L) in the barrel of the DNA column for
90 minutes. The column was washed from the bottom up with 85 mMNaCl in
0.01M K.sub.2 HPO.sub.4, 1 mM EDTA pH 6.8 (conductivity 8-9 mmho/cm) wash
buffer overnight. After a stable effluent A.sub.280 of 0.03 was reached,
the column was eluted from the bottom up with 330 mM NaCl in 0.01M K.sub.2
HPO.sub.4, 1 mM EDTA pH 6.8 elution buffer. The eluate was frozen at
-70.degree. C. in aliquots.
Enzyme and Inhibitory Activity Assay of ACT
Human neutrophil cathepsin G and bovine chymotrypsin esterolytic activity
were measured using succinyl-ala-ala-pro-phe-p-nitroanilide as substrate
(see DelMar, E. G., Largman, C., Broderick, J. W., et al.: A sensitive new
substrate for chymotrypsin, Anal. Biochem. 1979; 99:316-329). A Sequence
Information Sheet for the above substrate is included at the end of this
application. Inhibitory activity was measured by mixing a fixed quantity
of enzyme (cathepsin G/chymotrypsin) with varying amount of ACT. After
incubation for 5 minutes at 25.degree. C. the mixtures were assayed for
esterolytic activity. An extinction coefficient of 6.2 (1% solution, 280
mM) determined with purified inhibitor was used for the determination of
protein concentration (see Babul, J., Stellwagen, E.: Measurement of
protein concentration with interferences optics, Anal. Biochem. 1969;
28:216-221). Inhibitory specific activity was expressed as the ratio of
inhibitory activity per protein concentration.
Antigenic Activity of ACT Determined by RID and ELISA
The antigenic activity of the ACT was determined by a radial
immunodiffusion kit from The Binding Site (San Diego, Calif.). The
antigenic activity was also determined by enzyme linked immunosorbent
assay (ELISA). Purified ACT standard at a concentration range of 0.1-10
ng/ml or purified ACT preparation samples were captured with rabbit
antibody to ACT coated plates. A biotinylated rabbit antibody to ACT was
added to the plates, following incubation with peroxidase conjugated
streptavidin and substrate (tetramethyl benzidine) to detect the activity.
Antigenic specific activity was measured as the ratio of antigenic
activity per protein concentration.
Antigenic Activity of PI Determined by ELISA
The antigenic activity of PI was determined by enzyme linked immunosorbent
assay (ELISA). Goat anti-human .alpha..sub.1 -antiproteinase inhibitor
(Cappel, NC) is used as a coating antibody. Purified PI (Athens Research),
purified ACT preparation samples or lyophilized plasma standard of 1.3
mg/mL at a concentration range of 0.78-25 ng/ml were captured with goat
anti-human .alpha..sub.1 -antiproteinase inhibitor coated antibody.
Peroxidase conjugated goat anti-human .alpha..sub.1 -antiproteinase
inhibitor was added to the plates, following incubation with substrate
(tetramethyl benzidine) to detect the activity. Samples were compared
against a standard curve of absorbance vs. antigen concentration.
Enzyme and Inhibitory Activity Assay of AT-III (Antithrombin III)
This is a two-stage assay in which plasma standard or a concentration of
known AT-III activity and samples are diluted in buffer containing heparin
and incubated with bovine thrombin (Stage I). Chromogenic substrate S-2238
(H-D-phenylalanyl-L-pipecolyl-L-arginine-P-nitroaniline 2 HCl, Chromogenix
AB, Sweden) is then added, and hydrolyzed by any excess thrombin which has
not been inhibited by AT-III, released P-nitroanilide and absorbance at
405 nm is quantitated. AT-III concentration is inversely proportional to
the degree of hydrolysis (absorbance) by measuring the absorbance at 405
nm.
SDS PAGE and Western Blotting
Analytical electrophoresis was carried out on Tris-glycine 8-16%
polyacrylamide gradient slab gels (purchased from Novex System, San Diego,
Calif.) using the gel buffer system of Novex containing Tris-glycine, 0.1%
SDS pH 8.3. Purified ACT standard and sample preparations were prepared in
sample buffer containing Trisglycine, 2% SDS, 10% .beta.-mercaptoethanol
for 10 minutes in boiling water. Molecular weight electrophoresis
calibration kits from Novex Systems were used for molecular weight
determination. Proteins were detected by Coomassie Brilliant Blue G-250.
Western blotting was performed from gel to nitrocellulose membrane and
detected by rabbit antisera to human ACT and alkali phosphate conjugated
goat anti rabbit IgG.
HPLC Analysis
ACT standard from Athens Research and purified ACT from the scale-up
process were analyzed on TSK 3000 SW with 0.05M Na.sub.2 HPO.sub.4, 0.15M
NaCl pH 6.5 as the mobile phase.
RESULTS
To prepare human ACT, Cohn Fraction IV-1 paste suspension was employed as
starting material. Fraction IV-1 paste contains approximately the
following mg/g paste of three proteinase inhibitors: .alpha..sub.1
-proteinase inhibitor (PI)--30.0; ACT--5-10.0 determined by antigenic
activity ELISA and AT-III--5.0 measured by enzyme inhibitory activity. ACT
purification by PEG precipitation, S-Sepharose.RTM. and Q-Sepharose.RTM.
chromatography was unfavorable as there was no improvement in purity.
Ammonium sulfate fractionation, chelating column and Cibachrome Blue.RTM.
column for the purification process were unacceptable due to low yield and
low purity of ACT.
When employing Fraction IV-1 paste suspension (20733-91-1) in 0.01M Tris pH
6.5 at low conductivity (1.0-2.5 mmho/cm) on DEAE-Sepharose.RTM., more
than 91% of total protein was present in the 0.025M NaKPO.sub.4 pH 6.5
flow through fraction (20767-36-1 and -2) and 0.075M NaKPO.sub.4 pH 6.5
wash fraction (20767-36-3) (Table 1).
TABLE 1
______________________________________
DEAE-Sepharose Column (10 ml)
Specific
Total Yield % Activity
ACT of Total mg ACT/
Sample .mu.g ACT mg Protein
______________________________________
FIV-1 Paste Suspension (20733-
4,326 100 0.023
91-1)
0.025M NaKPO.sub.4 pH 6.5 Flow
257.2 6.0 0.004
thru (20767-36-1 & -2)
0.075M NaKPO.sub.4 pH 6.5 Wash
166.5 3.8 0.003
(20767-36-3)
0.1M NaKPO.sub.4 pH 6.5 Eluate
2,188.3 50.6 0.169
(20767-36-4 & -5)
0.2M NaKPO.sub.4 pH 6.5 Eluate
732 16.9 0.04
(20767-36-6)
______________________________________
More than 50% of ACT eluted at 0.1M NaKPO.sub.4 pH 6.5 (20767-36-4 and -5)
and there was a seven-fold increase in ACT specific activity. However,
loading Fraction IV-1 paste suspension in 0.01M K.sub.2 HPO.sub.4 pH 6.8
directly onto a DNA-cellulose column (5 ml) gave only 10% recovery yield
with a four to ten fold increase in purity after elution with 0.01M
K.sub.2 HPO.sub.4, 0.33M NaCl pH 6.8. Therefore, DEAE-Sepharose.RTM. is
the preferred first step for ACT prepared from Fraction IV-1 paste
suspension.
The following examples were carried out with Cohn Fraction IV-1 paste
suspension in 0.01M Tris pH 6.5 at conductivity 1.0-2.5 mmho/cm, followed
by DEAE-Sepharose.RTM. and DNA-cellulose chromatography.
EXAMPLE 1
ACT in Cohn Fraction IV-1 paste was suspended in 0.01M Tris pH 6.5 and
purified through a DEAE-Sepharose.RTM. column. The column was washed with
0.025M to 0.075M Sodium Phosphate (NaHPO.sub.4) pH 6.5, conductivity=1.9
to 5.3 mmho/cm buffer and 3.6% of the total ACT was eluted with 0.1M
Sodium Phosphate pH 6.5 (conductivity: 6.5 mmho/cm) (21864-4-C) and 46% of
the total ACT with three and a half fold increase in specific activity
(0.072 mg ACT/mg protein) eluted at 0.2M Sodium Phosphate pH 6.5
(conductivity 13.0 mmho/cm) (21864-4-D) (Table 2).
TABLE 2
______________________________________
Comparison of Elution Buffer System on
DEAE-Sepharose Column for ACT Purification
Specific
Total Activity
ACT Yield mg ACT/mg
Sample .mu.g % Protein
______________________________________
A.
FIV-1 Paste suspension
10,250 100 0.021
0.025M NaHPO.sub.4 pH 6.5 Flow
1,147 11.2 0.007
Thru (21864-4-A)
0.075M NaHPO.sub.4 pH 6.5 Wash
335 3.3 0.003
(21864-4-B)
0.1M NaHPO.sub.4 pH 6.5 Eluate
372 3.6 0.009
(21864-4-C)
0.2M NaHPO.sub.4 pH 6.5 Eluate
4,760 46.4 0.072
(21864-4-D)
B.
FIV-1 Paste Suspension DP
4,580 100 0.024
1279
0.025M NaKPO.sub.4 pH 6.5 Flow
457.6 10.0 0.009
Thru (21893-17A)
0.075M NaKPO.sub.4 pH 6.5 Wash
1,200.5 26.2 0.013
(21893-17B)
0.1M NaKPO.sub.4 pH 6.5 Eluate
2,059.6 45.0 0.134
(21893-17C)
0.2M NaKPO.sub.4 pH 6.5 Eluate
235.6 5.1 0.03
(21893-17D)
______________________________________
However, with 0.025M to 0,075M Sodium Potassium Phosphate (NaKPO.sub.4) pH
6.5, conductivity: 2.8 to 7.4 mmho/cm buffer wash, partially purified ACT
eluted at 0.1M NaKPO.sub.4 pH 6.5 (conductivity: 9-10 mmho/cm)
(21893-17-C) from DEAE-Sepharose.RTM. and gave 45% yield with 5.6 fold
increase in specific activity (0.134 mg ACT/mg protein) (Table 2). The
results in Table 2 indicate that employing the equilibrated buffer system
0.025M NaKPO.sub.4 pH 6.5 (conductivity: 2.8-3.0 mmho/cm) for
DEAE-Sepharose.RTM. and loading Cohn Fraction IV-1 paste suspension
(conductivity: 1.0-2.5 mmho/cm) on DEAE-Sepharose.RTM. column, remove
unwanted protein by washing with 0.075M NaKPO.sub.4 pH 6.5 (conductivity:
7.0-7.8 mmho/cm) and partially purified ACT (DEAE-eluate) elutes with 0.1M
NaKPO.sub.4 pH 6.5 (conductivity: 10 mmho/cm) for further purification
through a DNA-cellulose column.
EXAMPLE 2
When Cohn Fraction IV-1 paste suspension (DP 1279) with low antigenic
specific activity (0.024 mg ACT/mg protein) was used as starting material
for purification through DNA-cellulose (Pharmacia), there was a low yield
of ACT (17%), low purity (antigenic specific activity 0.12-0.32 mg ACT/mg
protein) and low ACT binding capacity (0.1-0.2 mg ACT/mL DNA-gel, Table
3).
TABLE 3
______________________________________
FIV-1 Paste Suspension on DNA-Cellulose Column (5 ml)
Spec.
Total Yield % Activity
ACT of Total mg ACT/
Sample .mu.g ACT mg Protein
______________________________________
FIV-1 Paste Suspension
4,580 100 0.024
(DP1279) (21893-20-1)
Flow Thru (21893-20-A)
1,775.2 38.8 0.012
0.085M NaCl Wash (21893-
356 7.8 0.017
20-B)
0.33M NaCl Eluate (21893-20-
765.6 16.7 0.324
33,34,35)
______________________________________
However, partially purified ACT in DEAE-eluate (20767-93-1, 21893-15-1)
with higher antigenic specific activity (0.16-0.25 mg ACT/mg protein) as
starting material resulted in higher yield (61.2-67.1%), higher purity
(antigenic specific activity 1.07-1.47 mg ACT/mg protein) and higher ACT
binding capacity (0.6-1.2 mg ACT/mL DNA-gel, Table 4). Based on these
data, DNA-cellulose is the second step for purified ACT prepared from
partially purified DEAE-eluate.
TABLE 4
______________________________________
DEAE Eluate on DNA-Cellulose Column
Total ACT Specific
Antigenic Yield Activity mg
Sample .mu.g % ACT/mg Protein
______________________________________
EXPERIMENT 1
Conc. of DEAE-Eluate
1,605.5 100 0.248
(20767-93-1)
Flow Thru (20767-93-A)
30.6 1.9 0.004
0.085M NaCl Wash
38.3 2.4 0.07
(20767-93-B)
0.33M NaCl Eluate
982 61.2 1.07
(20767-93-C&D)
EXPERIMENT 2
Conc. of DEAE-Eluate
4,230 100 0.164
(21893-15-1)
Flow thru (21882-29-A)
51.4 1.2 0.003
0.085 NaCl Wash
29.4 0.7 0.03
(21882-29-B)
0.33M NaCl Eluate
2,837.2 67.1 1.47
(21882-29-21,22,23)
______________________________________
EXAMPLE 3
Our overall preferred process for preparing ACT from plasma is shown in
FIG. 2. In a proposed scale-up process, 2 lots of partially purified ACT
prepared from Fraction IV-1 paste suspension (70 kg) at pH 6.5,
conductivity 1.0-2.0 mmho/cm, through DEAE-Sepharose.RTM.column (30 L)
resulted in a 17-34% yield at 0.1M NaKPO.sub.4 pH 6.5 eluate (conductivity
9.5-10.5 mmho/cm) with antigenic specific activity increased from 0.022 to
0.108-0.194 mg ACT/mg protein and a four to nine fold increase in purity
(Table 5).
TABLE 5
______________________________________
Prepare Purified ACT by Scale-up Process
Antigenic
Specific
Total ACT Activity
Antigenic Yield mg ACT/mg
Sample mg % Protein
______________________________________
EXPERIMENT 1
Step 1: DEAE-Sepharose
(30 L)
FIV-1 Paste Suspension
25,690 100 0.022
(21893-58-2)
DEAE Flow Thru 885 3.4 0.003
(21893-58-3)
DEAE Wash (21893-58-4)
4,865 18.9 0.015
DEAE Eluate (21893-58-5)
8,710 33.9 0.194
Step 2: DNA-Cellulose (3 L)
DF/UF Conc DEAE-Eluate
8,494 33.1 0.187
(21893-58-6)
DNA-Eluate (21869-35)
3,464 13.5 1.24
EXPERIMENT 2
Step 1: DEAE-Sepharose
(30 L)
FIV-1 Paste Suspension
31,150 100 0.025
(21882-65-1)
DEAE-Flow Thru 1,854 6.0 0.008
(21882-65-2)
DEAE-Wash (21882-65-3)
6,470 20.8 0.008
DEAE-Eluate (21882-65-4)
5,265 16.9 0.108
Step 2: DNA-Cellulose (3 L)
DF/UF Conc. DEAE-
4,939 15.9 0.094
Eluate (21882-65-5)
DNA-Eluate (21869-37)
2,142 6.9 1.24
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Insignificant loss of activity of ACT was obtained through the DF/UF step.
DEAE-eluate was further purified through DNA-cellulose column (3 L) and
gave overall yield of 6.9-13.5% with antigenic specific activity increased
from 0.108-0.194 to 1.24 mg ACT/mg protein. Two lots of DNA-eluate
(21869-35, 21869-37) were further diafiltered and ultrafiltered,
formulated in PBS buffer pH 7.0 at 10-20 mg/mL (21893-61-3 and 21882-68)
and stored at -70.degree. C. for further in vitro and in vivo studies.
Characterization of Purified ACT
A. Antigenic and Inhibitory Activity
Purified ACT (21893-61-3, 21882-68) prepared from Cohn Fraction IV-1 by
scale-up process including the in-house DNA-cellulose column resulted in
specific activity of >1 mg/mg total protein of inhibitory activity to both
human cathepsin G and to bovine chymotrypsin when compared to purified ACT
standard (freeze dried form) purchased from Athens Research, Atlanta, Ga.
(Table 6).
TABLE 6
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Antigenic and Inhibitory Activities of Purified ACT
ACT Activity (mg/mL)
Spec. Act. mg ACT/mg Protein
Protein Inhibitory Inhibitory
Conc Cathepsin Cathepsin
Purified ACT
(mg/mL)
Antigenic
G Chymotrypsin
Antigenic
G Chymotrypsin
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21893-61-3
21.7 26.2 24.3 -- 1.2 1.1 --
21882-68
9.7 9.6 12.1 14.1 0.99 1.36 1.45
ACT Standard
2.63 2.53 2.9 2.8 0.96 1.1 1.05
(Athens
Research)
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B. SDS PAGE and HPLC Analysis
When 5 .mu.g, 10 .mu.g, 20 .mu.g per lane was applied on 6-18% SDS-PAGE
under reducing conditions, ACT standard prepared from plasma and purified
ACT (21893-61-3) prepared from Fraction IV-1 paste had similar molecular
weight ranges (59.2-61.9 kD) and similar purity (.gtoreq.90%). By Western
blotting, ACT prepared from plasma and Fraction IV-1 paste could be
detected with anti-human ACT antibody, but there were no significant bands
detected in either preparations with either anti-.alpha..sub.1 -PI or
anti-AT-III antibody. Purified ACT (21893-61-3) contained trace amount of
degraded fragment (25.5 kD) which bound to anti-human ACT antibody in
Western blotting. HPLC analysis showed that purified ACT from both plasma
and Fraction IV-1 paste had similar retention times and more than 90%
purity.
C. IEF
ACT standard from Athens Research and purified ACT from Fraction IV-1 paste
(21893-61-3, 21882-68) were analyzed on Novex IEF (pH 3-10, Novex System,
San Diego, Calif.). Both ACT from plasma (see Laursen, I. and Lykkesfeldt,
A. E.: Purification and characterization of an .alpha..sub.1
-antichymotrypsin-like 66 kD protein from the human breast cancer cell
line, MCF-7, Biochem. Biophys. Acta. 1992; 1121:119-129) and from Fraction
IV-1 paste had similar pI's of around 3.8-4.3 compared with pI's of around
4.6-5.1 for AT-III and 4-5 for PI.
D. Specifications of Purified ACT (21882-68)
2.4 g of purified ACT (21882-68), 10 mg/ml in PBS buffer prepared from two
runs of the scale-up process has been characterized. The purified ACT has
.gtoreq.90% purity by IEF, SDS PAGE and HPLC, biological activity
comparable to ACT standard (purchased from Athens Research) and contains
0.7 endotoxin units per mg A.sub.280 protein by LAL assay (Table 7).
TABLE 7
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Characteristics of Purified ACT (21882-68)
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Volume 259 mL, 50 vials (5 mL/vial)
Characteristics
Appearance (Visual)
Clear, colorless solution
Protein Conc. (A.sub.280)
0.1% E1cm = 0.62 10.1 .+-. 0.4 mg/mL
PBS Buffer pH 7.1 .+-. 0.1
Identification
Isoelectric Focusing
IEF spectrotype about 90%
complies with ACT std.*
Purity
SDS PAGE (6-18%) - Reduced
.gtoreq.90% ACT std. with trace
amount of degraded
fragment (25.5 kD)
HPLC Area % of ACT std. .gtoreq.90%
Endotoxin (LAL) 0.7 EU per mg A.sub.280 protein
Biological activity (antigenic,
Comparable to ACT std.
inhibitory to human cathepsin G
and bovine chymotrypsin
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*Purchased from Athens Research
DISCUSSION
Cohn Fraction IV-1 paste contains a slightly higher concentration of ACT
(5-10 mg/g paste) than plasma (4-5 mg/g protein). Therefore, Cohn Fraction
IV-1 paste suspension fractionated from human plasma is the preferred
starting material for further purification. Human ACT has been purified to
homogeneity from human plasma pool by various procedures, such as ammonium
sulfate fractionation, Cibacron Blue.RTM. chromatography, SP-Sephadex.RTM.
or QAE-Sephadex.RTM., DNA-cellulose, and S-300 chromatography. However,
contrary to the published results of others, we found that ACT
purification with ammonium sulfate fractionation or direct DNA-cellulose
chromatography resulted in low yield and low purity when employing
Fraction IV-1 paste as starting material.
Prolastin.RTM., a commercially available plasma-derived PI preparation
(Miles Inc.) purified by PEG precipitation and DEAE-Sepharose.RTM. from
Fraction IV-1 paste suspension, is comprised of 600 .mu.g PI per mg
protein and a small amount of ACT, 29 .mu.g per mg protein. This
reinforces our findings that PEG precipitation of Fraction IV-1 paste
results in low yield and low purity of ACT. Another commercial plasma
protein preparation is antithrombin III (Thrombate.RTM., Miles Inc.)
prepared from Fraction IV-1 paste suspension by heparin-agarose
chromatography. Our studies showed that more than 91% of ACT did not bind
to the heparin-agarose column. This would imply that ACT has the potential
to be prepared as an AT-III side product.
Cohn Fraction IV-1 paste contains three major proteinase inhibitors; PI (30
mg/g paste), AT-III (5 mg/g paste) and ACT (5-10 mg/g paste). These
proteins have very similar pI values; 4.6-5.1 for AT-III, 4-5 for PI,
3.8-4.3 for ACT; and similar molecular weights: 58 kD for AT-III, 53 kD
for native PI, and 58-68 kD for native ACT. Optimal separation of ACT from
PI and AT-III from Cohn Fraction IV-1 paste was dependent on employing the
right kind of anion exchange resin with the right kind of buffer, pH and
ionic strength (conductivity) to remove PI and AT-III in the purified ACT
preparation. ACT purification from Cohn Fraction IV-1 paste suspension at
pH 6.5 and conductivity of 1-2.5 mmho/cm through DEAE-Sepharose.RTM. (but
not Q-Sepharose.RTM.), pre-equilibrated with 0.025M NaKPO.sub.4 pH 6.5
buffer (conductivity 2.5-3 mmho/cm), removed most PI at 0.075M NaKPO.sub.4
pH 6.5 (conductivity 7.4-7.8 mmho/cm) and ACT was eluted at 0.1M
NaKPO.sub.4 pH 6.5, 9-10.5 mmho/cm conductivity. The partially purified
ACT from DEAE-eluate containing AT-III and traces of PI was further
purified through DNA-cellulose. Most of the AT-III and traces of the PI
did not bind to DNA-cellulose when the column was washed with 0.01M
K.sub.2 HPO.sub.4 buffer, 0.085M NaCl pH 8.0 conductivity 8-9 mmho/cm, and
only activated ACT specifically absorbed to DNA-cellulose and eluted at
0.33M NaCl in 0.01M K.sub.2 HPO.sub.4 pH 6.8 (conductivity 24-25 mmho/cm).
Overall yield and purity of ACT following the DNA-cellulose column are
dependent upon purity of the ACT solution loaded onto the column.
DNA-cellulose removed unbound AT-III and PI when partially purified ACT
(DEAE-eluate) but not Cohn Fraction IV-1 paste suspension was loaded onto
the column. This resulted in highly purified ACT with specific activity of
>1 mg/mg protein of inhibitory activity to both human cathepsin G and to
bovine chymotrypsin.
Purified ACT prepared from Cohn Fraction IV-1 paste exhibits a similar
molecular weight range by SDS PAGE and HPLC analysis and similar
isoelectric points when compared with ACT standard prepared from human
plasma. The antigenically intact and active ACT can be purified from Cohn
Fraction IV-1 paste and further studies of its in vivo biological activity
are required to support its therapeutic use.
Given the above examples, it is thought variations will occur to those
skilled in the art. Accordingly, it is intended that the invention
disclosed here should be limited only by the following claims.
__________________________________________________________________________
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 1
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4
(B) TYPE: amino acid
(C) STRANDEDNESS: single strand
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(A) DESCRIPTION: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified peptide used for enzyme activity assay
(D) OTHER INFORMATION: Peptide is modified with n-terminal
succinyl group and c-terminal p-nitroanilide group
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Del Mar, E. G.
Largman, C.
Broderick, J. W.
(B) TITLE: A Sensitive New Substrate for Chymotrypsin
(C) JOURNAL: Analytical Biochemistry
(D) VOLUME: 99
(F) PAGES: 316-329
(G) DATE: 01-NOV-1979
(K) RELEVANT RESIDUES IN SEQ ID NO:1: FROM 1 TO 4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AlaAlaProPhe
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